A small number of genes in mammals are regulated by genomic imprinting, a process that results in unequal expression of the maternal and paternal alleles of certain genes. As a consequence, deleterious mutations or deletions in the single expressed allele of an imprinted gene will result in the absence of a functional gene product. In humans, disruptions in imprinting and imprinted genes account for the human genetic diseases Beckwith-Wiedemann Syndrome, Prader-Willi Syndrome and Angelman Syndrome and for cancers such as Wilms' tumor. The objective of this proposal is to investigate the mechanism by which parental identity of imprinted genes is established. The studies will employ the H19 gene, which is expressed from the maternally derived allele in mice and humans. Specifically, the role of a region differentially methylated at the mouse H19 locus will be studied. This differentially methylated domain (DMD) is located from -2 kb to -4 kb relative to the start of H19 transcription and is hypermethylated exclusively in male gametes and on the paternal allele throughout development, thereby fulfilling important criteria for the mark that distinguishes parental alleles of H19. The DMD is essential to imprint H19 transgenes as well as the endogenous H19 gene, and the linked and oppositely imprinted Igf2 gene. This proposal will investigate the role of the 2 kb DMD through the following experiments: (1) to determine if the DMD is sufficient for imprinting, hybrid transgenes that link the DMD to an H19 minigene or a heterologous gene and regulatory elements will be tested for imprinting in mice; (2) to determine whether specific CpG dinucleotides within the DMD are essential for imprinting, transgenes that harbor mutations of individual CpG dinucleotides will be examined for imprinting in mice; (3) to investigate whether the DMD is required for the establishment and/or maintenance of the genomic imprint, mice that harbor a deletion of the DMD at the endogenous locus will be examined for early imprinted expression; additionally, mice that harbor a conditional allele of the DMD deletion will be generated so that the DMD can be removed at a time after imprinted H19 expression is observed; (4) to determine when parental identity of H19 is acquired during gametogenesis, male germ cells will be studied to determine when the paternal-specific pattern of methylation is first imposed on the H19 gene. Knowledge of the sequences required for imprinting and the timing of imprint establishment is essential for the identification of the trans-acting factors that mediate genomic imprinting.